GPCRs mediate many of the cellular actions of neurotransmitters and hormones in the body as well as the therapeutic effects of almost half of the drugs sold by the pharmaceutical industry and have been important molecular targets for drug discovery. The sequencing of the Human Genome has revealed as many as 800 GPCR genes, most of which are orphans with not known ligands. These orphan GPCRs represent a large pool of unique molecular targets for the discovery of new therapeutics. We propose to develop a novel approach for HTS of drugs against orphan GPCRs employing the basic finding that agonist binding to GPCRs causes the translocation of B-arrestin from the cytosol to the GPCR. B arrestin trafficking is usually measured by confocal microscopy which is low throughput, expensive and not easily amenable for HTS. We propose to develop a simple B-arrestin trafficking assay that is easily amenable for HTS using our enzyme fragment complementation (EFC) technology that can detect the intracellular translocation of proteins. The assay is based on an extremely sensitive B galactosidase (B-gal) complementation technology that utilizes 2 genetically engineered fragments of E. coli B-gal. The larger fragment, Enzyme Acceptor (EA), contains a deletion near the amino terminus, while the smaller fragment, ProLabel, contains the amino-terminal sequence missing from EA. Alone, EA is inactive. However, in vitro it can spontaneously recombine with Pro-label to form an active enzyme that catalyzes the formation of a luminescent product that can be detected photometrically. The objective of this grant will be to use our EFC technology to measure B-arrestin translocation in response to GPCR activation as a highly sensitive drug discovery assay for orphan GPCRs and GPCRs in general. This technology could also be used to identify allosteric modulators of GPCRs which are a novel class of drugs that modify sensitivity and extent of GPCR activation without directly interacting with the ligand binding domains. Known allosteric modulators are ions and G proteins, which affect the affinity of GPCRs for agonists and drugs such as MPEP that antagonize transmission via some metabotrophic receptors without affecting agonist binding. Allosteric modulators can have novel therapeutic uses since they primarily act when the receptor is stimulated, thereby tempering transmitter and agonist activation of the receptor. In this grant, we will test whether the GPCR EFC assay developed can be employed as a sensitive assay to identify GPCR allosteric modulators to provide a totally novel avenue of GPCR drug discovery.